The three aims of this project are 1) to identify proteins that interact with STAMP, 2) to see if changes in Amax and PAA with STAMP can be dissociated, and 3) to search for other activities of STAMP. Aim 1 is being assessed by generating knock out (KO) mice for STAMP, preparing immortalized mouse embryo fibroblasts (MEFs) from the KO and wild type mice, and then running microarray analyses on KO and wild type MEF cells after treatment with steroid. After considerable time and effort by a commercial company (inGenious Targeting Laboratory), heterozygous mice containing one allele of a conditional KO (Cre/Lox) STAMP gene were obtained. The females were bred with male EIIA-Cre mice (from Dr. Heiner Westphal, NICHD/NIH) and the progeny mated to obtain heterozygous STAMP KO males and females. These heterozygots mice were mated, the embryos were geneotyped, and the appropriate MEF cells were prepared and immortalized by continuous growth. Nine endogenous genes were examined for GR-inducibility. After confirming that the KO MEF cells did indeed have &#8804;0.02% of the levels of STAMP mRNA in wild type MEF cells, mRNA for microarray analyses was prepared from cells that were treated with vehicle or steroid for 8 hr. High quality data were obtained for almost 3,000 genes, for which the level of expression changed by &#8805;1.5 fold after Dex treatment. These studies are on-going, with particular emphasis on the number and nature of those genes that display increased or decreased gene expression in the absence of STAMP, on the ability of STAMP to affect Amax and PAA independently (as we observed for the GILZ gene in PBMCs), and on the identity of cellular pathways that might be especially sensitive to the levels of STAMP. Aim 3 asks whether STAMP mediates any other actions. We recently reported that changing the level of STAMP (by overexpression of transfected plasmid or by reducing the endogenous protein with siRNAs) alters the growth behavior of cells in a cell-selective and steroid-independent manner. Also, screening of a variety of cancers uncovered a strong positive correlation between the presence of ovarian cancers and elevated STAMP mRNA levels. The results of the above microarrays are being carefully studied for indications of how STAMP might influence cell growth and/or ovarian tumor formation. In summary, we have prepared MEF cells from STAMP KO mice. These cells are invaluable in determining the effects of a known modulatory factor both on the parameters of GR-regulated induction (EC50, PAA, and Amax) and on apparent receptor-independent effects for cell growth and tumor formation. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.